Synthesis and Systematic Investigation of Lepidiline A and Its Gold(I), Silver(I), and Copper(I) Complexes Using In Vitro Cancer Models and Multipotent Stem Cells

The imidazole alkaloid lepidiline A from the root of Lepidium meyenii has a moderate to low in vitro anticancer effect. Our aim was to extend cytotoxicity investigations against a panel of cancer cells, including multidrug-resistant cancer cells, and multipotent stem cells. Lepidiline A is a N-heterocyclic carbene precursor, therefore a suitable ligand source for metal complexes. Thus, we synthesized lepidiline A and its copper(I), gold(I), and silver(I) complexes and tested them against ovarian, gastrointestinal, breast, and uterine cancer cells and bone marrow-derived and adipose-derived mesenchymal stem cells. Lepidiline A and its copper complex demonstrated moderate cytotoxicity, while silver and gold complexes exhibited significantly enhanced and consistent cytotoxicity against both cancer and stem cell lines. ABCB1 in the multidrug-resistant uterine sarcoma line conferred significant resistance against lepidiline A and the copper-lepidiline A complex, but not against the silver and gold complexes. Our results indicate that only the copper complex induced a significant and universal increase in the production of reactive oxygen species within cells. In summary, binding of metal ions to lepidiline A results in enhanced cytotoxicity with the nature of the metal ion playing a critical role in determining its properties.


Table of contents
Table S1.Cytotoxicity of the tested compounds Table S2.Binding energy release Table S3-S5.Elemental analysis of Cu-LA, Ag-LA and Au-LA Table S6.Experimental details of X-ray diffraction study Table S7.Stability in solution of Cu-LA, Ag-LA and Au-LA  S1.Cytotoxicity of the tested compounds IC 50 values and the respective standard deviations (SD) calculated from pIC 50 values for all the tested compounds against ovarian cancer cell lines (IGROV-1, OVC-3, OVC-8), an upper gastrointestinal cancer line (OVC-5), breast cancer lines (MDA-MB-231, MCF-7, T-47D), uterine sarcoma lines (Mes-Sa, Mes-Sa/B1, Mes-Sa/Dx5), adipose tissue derived multipotent stem cell line (AD-MSC), bone-derived multipotent cell lines (BM-MSC, immortalized BM-MSC).LA: lepidiline A, Cu-LA: copper(I)-lepidiline A, Ag-LA: silver(I)-lepidiline A, Au-LA: gold(I)-lepidiline A. In a crystal structure harboring only NADP (1QYV), when SUB is physiologically available, E1, E2 and LA were all docked to SUB.The predicted free energy release by the binding of estrogens triggered 9.1 kcal/mol and 9.0 kcal/mol for E1 and E2, respectively, which is an order of magnitude greater than the free energy release upon LA binding (-8.2 kcal/mol).After we removed E2 from a crystal structure containing both NADPH and E2 (1A27), both E1 and E2 docked in SUB with high affinity (-9.2 kcal/mol and -9.4 kcal/mol, respectively), while LA docked with -7.5 kcal/mol).

LA
Although, compared to E1 and E2 docking, the estimated binding energy release was significantly lower in case of LA docking, since LA's concentration is approximately 3-order of magnitude higher at cytotoxic concentrations compared to estrogens [3], it might occupy the binding pockets, causing HSD17B1 inhibition, as binding energy release is proportional to the logarithm of concentration (∆G = -RT ln(c)).Geometric parameters of distances, angles and torsion angles (Å, º) for Au-LA:

Figure S1 .
Figure S1.Docking of estrone and lepidiline A Figure S2-S5. 1 H and 13 C NMR Spectra Figure S6.X-ray structures and geometric parameters of Ag-LA Figure S7.X-ray structures and geometric parameters of Au-LA

Figure S1 .
Figure S1.Docking of estrone (yellow) and lepidiline A (pink) in the PDB structure 1QYV of human HSD17B1 in far view and close view, visualized by PyMOL.Surface has 20% transparency to see cartoon representation.

Figure S7 .
Figure S7.X-ray structures and geometric parameters of Au-LA.A) ORTEP view of Au-LA complex at 50% probability level with partial numbering scheme.C31 and C14 are refined isotropically.B) Packing diagram of Au-LA with 3.9 Å of gold-gold distance, and 5.2 Å distance of Au-Cl with the neighboring molecule, indicating that the chloride ion is not bridging.The dimers are connected by C-H..Cl hydrogen bonds through a phenyl proton with H..Cl distance of 2.9 Å. A) B)

Table S4 :
Results of the elemental analytic measurements for Ag-LA

Table S5 :
Results of the elemental analytic measurements for Au-LA

Table S6 .
Experimental details of X-ray diffraction of Ag-LA and Au-LA Suitable crystals were fixed on a Mitegen loop using high viscosity oil.Data were collected at room temperature using a Bruker-D8 Venture diffractometer equipped with INCOATEC IμS 3.0 dual (Cu and Mo) sealed tube microsources and Photon 200 Charge-integrating Pixel Array detector, MoK ( = 0.71073 Å) radiation was used.Data collection and integration was performed using the APEX4 software[4].Data reduction and multi-scan absorption correction was applied[5].The structure could be easily solved using direct methods and refined on F 2 using SHELXL program [6] incorporated into the APEX4 suite.Refinement was performed anisotropically for non-hydrogen atoms except two phenyl carbon atoms in Au-LA as turning them anisotropically they became non positive definit.Hydrogen atoms were placed into geometric positions.Tables were extracted from the edited CIF file using publCIF[7].The PLATON program was used for crystallographic calculations.

Table S7 .
Stability of Cu-LA, Ag-LA and Au-LA in serum-free culture medium.Values indicate AUC, measured by HPLC-UV.